Tumor Necrosis Factor (TNF) and interleukin-1 (IL-1) are cytokines that have been implicated in a wide range of biological processes, including inflammation. The recruitment of immune cells to sites of injury involves the concerted interactions of a large number of soluble mediators. Several cytokines appear to play key roles in these processes, particularly IL-1 and TNF. Both cytokines are derived from mononuclear cells and macrophages, along with other cell types. Physiologically, they produce many of the same proinflammatory responses, including fever, sleep and anorexia, mobilization and activation of polymorphonuclear leukocytes, induction of cyclooxygenase and lipoxygenase enzymes, increase in adhesion molecule expression, activation of B-cells, T-cells and natural killer cells, and stimulation of production of other cytokines. Other actions include a contribution to the tissue degeneration seen in chronic inflammatory conditions, such as stimulation of fibroblast proliferation, induction of collagenase, etc. They have also been implicated in the process of bone resorption and adipose tissue regulation. Thus, these cytokines play key roles in a large number of pathological conditions, including rheumatoid arthritis, septic shock, inflammatory bowel disease, bone mass loss, cancer, dermal sensitization disorders, diabetes, obesity and neurological conditions such as ischemic stroke, closed-head injuries, etc.
Cytokines trigger a variety of changes in gene expression in their target cells by binding and activating their respective cognate receptors. Receptor activation sets in motion certain biochemical events, including the activation of otherwise latent transcription factors. Members of the NF-κB Rel family of transcription factors represent some of the most prominent of these transcription factors, having been implicated in the regulation of genes involved in inflammation, cell proliferation, apoptosis, and several other basic cellular functions (Verma et al. Genes Dev. 9, 2723 (1995); Baichwal & Baeuerle, Curr. Biol. 7, 94 (1997)).
The best studied member of this family of transcription factors is NF-κB, which generally exists in cells as a heterodimer of two proteins: p50 (NF-κB1) and p65 (RelA), although homodimers of these individual components are also possible (Baeuerle and Baltimore, Cell, 53, 211 (1988); Baeuerle and Henkel, Annu. Rev. Immunol. 12, 141 (1994)). NF-κB, in its inactive form, resides in the cytoplasm of cells. In response to various types of stimuli, such as proinflammatory cytokines (e.g., TNF and IL-1), ultraviolet irradiation and viral infection (Verma, 1995; Baichwal, 1997; Cao et al. Science, 271, 1128 (1996)) NF-κB migrates to the nucleus.
In its inactive state, the NF-κB heterodimer is held in the cytoplasm by association with inhibitory IκB proteins. Recently, the three-dimensional structure of a NF-κB/IκB ternary complex has been solved (Huxford et al. Cell, 95, 759 (1998); Jacobs et al. Cell, 95, 749 (1998)). When cells are treated with the appropriate stimuli, such as IL-1 or TNF, intracellular signal transduction pathways are activated that lead to the eventual phosphorylation of IκB proteins on two specific residues (serines 32 and 36 in IκBα, serines 19 and 23 in IκBβ). Mutation of one or both serine residues renders IκB resistant to cytokine-induced phosphorylation. This signal-induced phosphorylation targets IκB for ubiquitination and proteosome-mediated degradation, allowing nuclear translocation of NF-κB (Thanos and Maniatis, Cell, 80, 529 (1995)). The only regulated step in the IκB degradation pathway is the phosphorylation of IκB by IkB kinases (IKK) (Yaron et al. EMBO J. 16, 6486 (1997)).
Several intermediate steps in the TNF- and IL-1-activated signaling pathways that result in IκB phosphorylation have been elucidated in recent years. Both pathways appear to merge at the level of the protein kinase NIK (NF-κB-inducing kinase) (Malinin et al. Nature, 385, 540 (1997); Song et al. Proc. Natl. Acad. Sci. USA, 94, 9792 (1997)). Similarly, the protein kinases MEKK1 and MLK3 have been implicated in the induction of IKK activity (Lee et al. Proc. Natl. Acad. Sci. USA. 95, 9319 (1998); Hehner et al. Mol. Cell. Biol. 20, 2556 (2000)). While the specific details remain somewhat unclear regarding how these or other intermediate proteins may interact with and/or stimulate IKK activity in cells, significant progress has been made in elucidating the enzymes responsible for IkB phosphorylation.
Two IKK enzymes, generally referred to as IKKα (or IKK-1) and IKK β (or IKK-2) (Woronicz et al. Science, 278, 866 (1997); Zandi et al. Cell, 91, 243 (1997); Mercurio et al. Science, 278, 860 (1997)) have been discovered. Both forms of IKK can exist as homodimers and as IKKα/IKK β heterodimers. Another recently discovered component of the IκB kinase complex is a regulatory protein, known as IKK-gamma or NEMO (NF-κB-Essential Modulator) (Rothwarf et al. Nature, 395, 297 (1998)). NEMO does not contain a catalytic domain, and thus it appears to have no direct kinase activity and it probably serves a regulatory function. Existing data suggest that the predominant form of IKK in cells is an IKKα/IKK β heterodimer associated with either a dimer or a trimer of NEMO (Rothwarf et al. Nature 395, 297 (1998)).
Biochemical and molecular biology experiments have clearly identified IKKα and IKKβ as the most likely mediators of TNF- and IL-1-induced IκB phosphorylation and degradation, which results in NF-κB activation and upregulation of families of genes involved in inflammatory processes (Woronicz et al. Science (1997); Karin, Oncogene 18, 6867 (1999); Karin, J. Biol. Chem. 274, 27339 (1999)). These kinases have also been identified as components of CD40 ligand-induced signaling. IKKα and IKKβ have very similar primary structures, displaying more than 50% overall sequence identity. In the kinase domain, their sequences are 65% identical.
Based on our present understanding of the critical role played by TNF and IL-1 in the wide array of pathological conditions described above, and the involvement of IKKα and IKKβ in the signal transduction of both cytokines, the discovery of small molecules that potently and selectively inhibit either of these kinases would result in a major advancement in the therapy of those conditions. In this application we describe novel compounds which display such desirable activity.